Hierarchical_Clustering.m and BasalandWingNBPKAnalysis.m are introduced into a paper in the review stage - this ReadMe will be updated once it has been published

Prior versions of the code and processes have been used within these articles:

Azzari NA, Segars KL, Rapaka S, Kushimi L, Rich CB, Trinkaus-Randall V. Aberrations in Cell Signaling Quantified in Diabetic Murine Globes after Injury. Cells. 2023 Dec 21;13(1):26. doi: 10.3390/cells13010026. PMID: 38201230; PMCID: PMC10778404.

Segars KL, Azzari NA, Gomez S, Machen C, Rich CB, Trinkaus-Randall V. Age Dependent Changes in Corneal Epithelial Cell Signaling. Front Cell Dev Biol. 2022 May 5;10:886721. doi: 10.3389/fcell.2022.886721. PMID: 35602595; PMCID: PMC9117764.

Lee Y, Kim MT, Rhodes G, Sack K, Son SJ, Rich CB, Kolachalama VB, Gabel CV, Trinkaus-Randall V. Sustained Ca2+ mobilizations: A quantitative approach to predict their importance in cell-cell communication and wound healing. PLoS One. 2019 Apr 24;14(4):e0213422. doi: 10.1371/journal.pone.0213422. PMID: 31017899; PMCID: PMC6481807.

Lee YK, Segars KL, Trinkaus-Randall V. Multiple Imaging Modalities for Cell-Cell Communication via Calcium Mobilizations in Corneal Epithelial Cells. Methods Mol Biol. 2021;2346:11-20. doi: 10.1007/7651_2020_329. PMID: 33159251; PMCID: PMC8787922.

		Using a photo editor software to convert image set/video file into a .avi
	 	Name the Folder var with the files folder
	  	Name the name var with the files name with the .avi removed 
	   	Run code

User is importing excel files generated from data directly instead of allowing the script to identify cells and pull intensity data itself
This is done during instances of poor identification of cells from the Code such as with tissue and ex vivo analysis
The user has to create the intensity file and centroid file themselves, 

   		This lab has used ImageJ/FUJI for this process taking advantage of the Region of Interest (ROI) tool 
     		and the measure and multi measure tools within the ROI manager
       		Position data was created with centroid data
	 	Intensity data was created with mean gray value
   			Intensity data has to be generated only using the Calcium indicator Channel
       		This creates data sets that can be generated and copied into Microsoft excel
	 		make sure to remove the cell count variables in the first column in the centroid data
    			and make sure to remove the time point data from the intensity data generated in multi measure

   Name the Folder var with the files folder.
   Name the position data var using a string with the file type extension removed - posfilename.
   Name the intensity data var using a string with the file type extension removed - intfilename.
   The var 'name' is used for the creation of data sets via the code to create a basic for these file name.
   Run code.

Skp - is the number of frames checked for nearby signaling activity/propagation of calcium events - it is set to four frames by default

percent - the percentage of the Max Mean intensity required for an intensity change to reach before it is classified as a detected event
	The default for cell culture is 40% (.4)
 	The default for ex vivo/tissue is 30% (.3)

NxtCeldis - the max pixel distance required between centroids for cells to be classified as nearby
	ex if distance between two centroids is beyond this value - they are not nearby each other 
 		and similar temporal relation between cellular activity cannot be due to propagation
   	If those same two centroids where within the distance/pixel value they are nearby each other
    		and the similar temporal cellular activity could be due to propagation of signal

mnsig - mean signaling of all identified cell
totPK - total signaling
NBPK - total nearby signaling --- cell to cell communication?
prbNB - probability a signal was nearby signal
rate - rate of signaling by identified cells

This code analyzes the total active time of identified cells and determines if they have low or high activity and everything in between
This code returns a dendrogram and a Histogram graphs to display these differences
	The code also generated binarized intensity files generated from the intensity files created or inputted into the code

This code is used to analyze the propagation of calcium signaling events between different layers of tissue epithelium/different videos taken from the same tissue at different z layers
This code works in a similar manner to GMFCWMDB_Pre_made_Int_and_Pos and Pkcorr, it required premade centroid and intensity excel files
It then uses that centroid data and intensity data to determine if cells had detected events and propagated events within their layers
Then it compared those two data sets with the centroid data between layers/files to determine if there was propagated events between the two different data sets
	This used altered NxtCeldis values because of the different sizes of cells used by this lab, Wing cells are larger and have a large NxtCeldis value, 
 	While basal cells are smaller then have a smaller NxtCeldis then default values described above

  	var that have to be inputted are:	
   	folder = '';
	wingposfilename = '';
	wingintfilename = '';
	basalposfilename = '';
	basalintfilename = '';

outputs are as stated:
	wingNcell	Number of wing cells
	totwingPK	total wing cell cellular signaling
	wingNBPK	Number of wing cell total nearby activity - signaling propagation within data set

	basalNcell	number of basal cells
	totbasalPK	number of basal cell signaling
	basalNBPK	number of basal cell signal propagation 

   	WtBNBPK		Wing to Basal NBPK
 	BtWNBPK		Basal to Wing NBPK
	TotalNBPK	Total cross layer/data set NBPK

	Scripts have been combined into get_marked_frames_modified_combined_with_main_driver_Bounds.m 
	this version only requires the .avi file to be uploaded once rather than twice across two separate scripts
	
	Need to have get_marked_frames_modified.m and main_driver_Bounds.m open within a 2016a MATLAB.
	
	Within get_marked_frames_modified.m change vid variable to name of .avi file.
		.avi file is of desired cell image/time series
		series is separated into its channels
		only Ca signaling channel is required
		save file as an .avi file - can be edit so it only reads certain bits of series
			edit video/series before placed into code - causes issues during process if not done this way
		name variable vid in line 2 as name of desired .avi file
		make sure .avi file is within MATLAB pathway
		pick start frame ----- 1
		create pos excel graph with code
		
	Within main_driver_bounds.m variable name on line 10 us = 'name of avi'
		run code
		frame_rate is one
		start_frame is one
		end_frame is total frames - 1
			i.e. total is 900 frames enter 899 frames
		name int excel file being created
		recall pos excel file from get_marked_frames_modified

	
	Save the cell identified figure 
	Save the hot spot/intensity graphs
	other graphs/figures are unnecessary for data analysis